PROJECT SUMMARY Human papillomavirus (HPV)-driven cancers are common and lethal. There are no biomarker-selected, molecular targeted therapies for HPV+ cancers. Most of those who progress after initial therapy with radiation and chemotherapy die within 3 years, demonstrating a significant translational knowledge gap and an unmet clinical need. Anti-PD1 immune checkpoint therapy (ICT) is effective for recurrent HPV+ cancers, but with response rates of <20% and a one-year survival rate of <40%. To address the unmet need for biomarker- driven therapy for HPV+ cancers, we tested the efficacy of 721 unique drugs in 16 HPV+ and 17 matched HPV-negative cell lines and identified Aurora kinase inhibitors as more effective in HPV+ than HPV negative cancers. We demonstrated that Aurora kinase inhibition leads to apoptosis in HPV+ cell lines in vitro and reduced the growth of an HPV+ HNSCC patient-derived xenograft tumor in vivo. HPV+ cancer cells may be sensitive to Aurora inhibition because of their low Rb expression. RB1 loss and Aurora inhibition are synthetically lethal in a variety of cancer cell lines and preclinical mouse models. Although the mechanism underlying this synthetic lethality is unknown, the fact that multiple mitotic genes were identified in screens suggests that Rb's roles in mitosis and genomic stability are a central part of this mechanism. HPV+ cancers may rely on mitotic kinases such as Aurora to maintain mitotic fidelity. We hypothesize that Aurora kinase inhibition results in cell death in HPV+ cancers due to Rb loss-induced genomic instability. Furthermore, we hypothesize that this cancer cell death will stimulate the cGAS/STING pathway producing type I interferons and the resulting immunogenic cell death will lead to host T-cell engagement and increased sensitivity to ICT. This second hypothesis is crucial because cancers nearly always develop resistance to even highly effective targeted therapies, limiting their long-term benefits. In contrast, ICT results in durable responses in some patients, making it imperative to seek strategies that enhance the efficacy of ICT. To meet our long-term goal of improving cure rates for those with HPV+ cancer, we propose mechanistic studies to elucidate the role of Rb loss-induced genomic instability in Aurora kinase inhibition mediated cancer cell death (Aim 1); in vivo experiments with the combination of Aurora kinase inhibition and ICT in HPV+ murine model (Aim 2); and to leverage tissue from a clinical trial to dentify biomarkers predicting response to the combination of immune checkpoint and Aurora kinase inhibition in patients with recurrent HPV+ cancers. Our proposed research will have a positive impact because it will address an important problem, the lack of curative therapy for recurrent HPV+ cancers, and may shift current clinical practice paradigms for these cancers by identifying rational ICT and targeted drug combinations.